Atrial fibrillation (AF), defined by disorganized atrial cardiac rhythm, is the most prevalent cardiac arrhythmia worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with AF risk, including the cardiogenic transcription factor genes TBX5, GATA4, and NKX2-5. We report that Tbx5 and Gata4 interact with opposite signs for atrial rhythm controls compared with cardiac development. Using mouse genetics, we found that AF pathophysiology caused by Tbx5 haploinsufficiency, including atrial arrhythmia susceptibility, prolonged action potential duration, and ectopic cardiomyocyte depolarizations, were all rescued by Gata4 haploinsufficiency. In contrast, Nkx2-5 haploinsufficiency showed no combinatorial effect. The molecular basis of the TBX5/GATA4 interaction included normalization of intra-cardiomyocyte calcium flux and expression of calcium channel genes Atp2a2 and Ryr2. Furthermore, GATA4 and TBX5 showed antagonistic interactions on an Ryr2 enhancer. Atrial rhythm instability caused by Tbx5 haploinsufficiency was rescued by a decreased dose of phospholamban, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, consistent with a role for decreased sarcoplasmic reticulum calcium flux in Tbx5-dependent AF susceptibility. This work defines a link between Tbx5 dose, sarcoplasmic reticulum calcium flux, and AF propensity. The unexpected interactions between Tbx5 and Gata4 in atrial rhythm control suggest that evaluating specific interactions between genetic risk loci will be necessary for ascertaining personalized risk from genetic association data.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, affecting more than 7 million Americans and 33 million people worldwide (1). AF is characterized by an irregular pattern of atrial depolarization, resulting in rapid and disorganized atrial conduction and lack of effective atrial chamber contraction. The rhythm abnormality in patients with AF manifests with circulatory deficits and systemic thromboembolism that greatly increase morbidity and mortality. Because age is an independent risk factor for AF, its prevalence is expected to rise significantly as the population ages. AF has become a major clinical and economic burden, owing to the limitations and side effects associated with current AF therapies. Although AF most often manifests in the context of pre-existing cardiac pathologies, such as hypertension and cardiomyopathy, idiopathic or lone AF forms have indicated a heritable component (2). Genome-wide association studies (GWAS) have to date identified more than 100 AF-associated loci, including many transcription factor (TF) loci, suggesting that transcriptional control of atrial rhythm is an important mediator of AF risk (3, 4).

Recent work has illuminated a role for abnormal cardiomyocyte calcium (Ca2+) handling in the cellular pathophysiology of AF. The current paradigm of AF causation describes ectopic (triggered) atrial activity, mediated by early and delayed afterdepolarization (EAD and DAD) events and a fibrillogenic substrate that propagates the abnormal triggers causing arrhythmia. EADs and DADs have been associated with abnormal cardiomyocyte Ca2+ handling, including RYR2 dysfunction, reduced sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) (encoded by ATP2A2) activity, and/or increased Na+/Ca2+ exchanger (NCX) activity (5–9). It remains to be defined how alterations of RYR2, ATP2A2, or NCX expression in AF models contribute to AF risk.

GWAS and familial inheritance have implicated transcription factor genes in AF pathogenesis, including the cardiogenic transcription factors (TFs) TBX5, GATA4, and NKX2-5 (2, 10). Autosomal dominant mutations in the T-box TF TBX5 cause Holt-Oram syndrome, characterized by upper limb malformations, congenital heart defects, cardiac conduction system abnormalities, and increased AF risk (11, 12). In addition, GWAS has identified common risk variants associated with PR interval and increased AF susceptibility in intergenic or intronic regions of TBX5 (13–16). Adult-specific Tbx5 deletion in mice leads to spontaneous and sustained AF, characterized by slowed conduction and decrements in the expression of ion channels linked to AF (17). TBX5 drives the atrial expression of Pitx2, and TBX5 and PITX2 comodulate the expression of cardiac rhythm effector genes, including Ryr2 and Atp2a2. These findings indicated that interactions between TBX5 and PITX2 provide tight control of an atrial rhythm gene regulatory network and that perturbation of this network triggered AF susceptibility (17). This example suggested that cardiac TFs implicated in AF by genetic association may coregulate a gene regulatory network for atrial rhythm homeostasis. GATA4 and NKX2-5 are particularly relevant candidates, given that they both interact physically and genetically with TBX5 during cardiac development. GATA4, a zinc finger transcription factor, plays critical roles in heart development and cardiomyocyte differentiation (18–20). Several GATA4 loss-of-function mutations have been reported to underlie AF susceptibility in humans (10, 21–23). GATA4 is highly expressed in adult cardiomyocytes; however, its specific role in atrial cardiac rhythm has not been investigated. NKX2-5, a homeodomain containing TF has been implicated in AF by GWAS and family studies (13, 24–26). Common variants associated with PR prolongation, a marker for increased AF risk, have also been identified close to NKX2-5 by GWAS (13). Heterozygous mutations in NKX2-5 are associated with a spectrum of congenital heart diseases (CHDs) in humans and mice (27–31). NKX2-5 also has been shown to regulate a number of target genes involved in the cardiomyocyte action potential, suggesting that it may play a direct role in AF predisposition (32).

Combinatorial interactions between TBX5, GATA4, and NKX2-5 are critical for heart development. TBX5, GATA4, and NKX2-5 physically interact, and CHD-causing (but not CHD-sparing) mutations in TBX5 abrogate these interactions (33). Mutations in GATA4 that disrupt transcriptional cooperativity with TBX5 result in CHD and impaired cardiac gene expression, leading to aberrant chromatin states and gene expression (33, 34). Tbx5/Gata4 double-heterozygous mice develop cardiac defects that are more severe than Tbx5 or Gata4 haploinsufficient mice alone, providing evidence for a cooperative interaction (35). TBX5, NKX2-5, and GATA4 synergistically activate multiple cardiac enhancers and promoters (33, 36–45). Cooperative interactions between TBX5, NKX2-5, and GATA4 on cardiac gene expression may rely on interdependent binding of these factors genome-wide, enabling co-regulation of the cardiac differentiation program (46). Removal of Tbx5 or Nkx2-5 in mouse embryonic stem cell cardiac differentiations or GATA4 in human induced pluripotent stem cardiac differentiations resulted in inappropriate distribution of the other TFs to lineage-inappropriate sites, inducing ectopic gene regulation (34, 46). This paradigm suggested that heterotypic interactions between these cardiogenic TFs may influence atrial rhythm control.

We investigated the combinatorial genetic interactions between the cardiogenic transcription factors Tbx5, Gata4, and/or Nkx2-5 in murine atrial rhythm control. We hypothesized that adult-specific combined Tbx5, Gata4, or Nkx2-5 haploinsufficiency may alter AF susceptibility and illuminate AF pathophysiology. Surprisingly, we found that Gata4 haploinsufficiency rescued the cardiac rhythm, cardiomyocyte electrophysiology, and molecular defects caused by Tbx5 haploinsufficiency. In contrast, introducing Nkx2-5 haploinsufficiency had no observed effect on the penetrance or severity of defects caused by Tbx5 haploinsufficiency alone. Gata4 haploinsufficiency rescued the calcium-handling defects and Ryr2 and Atp2a2 gene expression deficits caused by Tbx5 haploinsufficiency. We found that GATA4 negatively modulated TBX5 activation of a cis-regulatory element at Ryr2. This observation indicated that GATA4 could repress TBX5-dependent transcriptional activation in some contexts and provided a molecular model for the rescue of Tbx5 haploinsufficiency by Gata4 haploinsufficiency. We tested the hypothesis that rescue of Tbx5 haploinsufficiency was mediated by rescue of calcium homeostasis. Introduction of haploinsufficiency for phospholamban (Pln), a SERCA inhibitor, into Tbx5-haploinsufficient mice caused normalization of cardiac rhythm and rescue of SERCA activity. Rescue of the Tbx5-haploinsufficient phenotype by Gata4 deficiency illuminated a TF genetic interaction in atrial rhythm control and provided a molecular model for transcriptional control of cardiomyocyte calcium flux as a central component of atrial rhythm homeostasis.

We previously demonstrated that TBX5 drives atrial expression of Pitx2 and that TBX5 and PITX2 oppositely modulate the expression of cardiac rhythm effector genes, including Ryr2 and Atp2a2 (17). This finding suggested that the rescue of Tbx5 haploinsufficiency by Gata4 haploinsufficiency could occur through diminished Pitx2 levels. Pitx2 mRNA expression remained unchanged in Tbx5fl/+;R26CreERT2 and Gata4fl/+;R26CreERT2 mice 2 weeks after TM treatment, as previously described (P = 0.7049 Tbx5fl/+;R26CreERT2 compared with R26CreERT2 and P = 0.7261 Gata4fl/+;R26CreERT2 compared with R26CreERT2) (Figure 3B and ref. 17). Levels of Pitx2 mRNA were slightly higher in Gata4fl/+;Tbx5fl/+;R26CreERT2 mice compared with WT littermates (P = 0.0117); however, this increase was not significant compared with Tbx5fl/+;R26CreERT2 or Gata4fl/+;R26CreERT2 mice (P = 0.1208 vs Tbx5fl/+;R26CreERT2 and P = 0.1135 vs Gata4fl/+;R26CreERT2 mice, respectively). This observation suggested that the rescue of Tbx5 haploinsufficiency by Gata4 haploinsufficiency was not mediated by a reduction of Pitx2 expression.

We hypothesized that TBX5 and GATA4 directly coregulate expression of Atp2a2 and Ryr2. To test this hypothesis, we defined regions with overlapping chromatin occupancy for both TBX5 and GATA4 from published ChIP datasets (Figure 3, C and D, Supplemental Figures 6 and 7, and ref. 53). Candidate cis-regulatory elements (CREs) were refined by open chromatin regions from the HL-1 cardiomyocyte ATAC-seq dataset (54). We functionally interrogated these CREs for enhancer activity in the presence of TBX5 and/or GATA4. The Atp2a2 enhancer (mm9 Chr5: 122970476-122971591) demonstrated activation in response to TBX5 expression in human embryonic kidney (HEK) 293T cells, as previously described (Figure 3E and ref. 54). GATA4 similarly activated the enhancer and coexpression of TBX5, and GATA4 had no additive or synergistic effects by in vitro luciferase reporter assay in HEK293T cells (Figure 3E). In contrast, the Ryr2 enhancer was activated by TBX5 (P = 0.0013) but not GATA4 alone (P = 0.5923) in HEK293T cells (Figure 3F). Remarkably, co-expression of GATA4 suppressed TBX5-dependent transactivation of this Ryr2 enhancer (P = 0.0681 compared with TBX5 alone), suggesting antagonistic interactions of TBX5 and GATA4 on this CRE. We further tested the Ryr2 CRE in HL-1 cardiomyocytes, which possesses expression of cardiac transcription factors, including TBX5 and GATA4 (55). This enhancer previously demonstrated TBX5-dependent activity in HL-1 cells, and consistently, this enhancer alone showed activation in HL-1 cardiomyocytes (Figure 3G and ref. 54). Interestingly, mutation of the 4 GATA binding motifs within this enhancer caused an increase of enhancer activity in HL-1 cardiomyocytes (P = 0.0010 vs WT enhancer) (Figure 3G). These observations indicated that GATA4 represses TBX5-dependent activity of the Ryr2 enhancer.

We report that TBX5 and GATA4, both implicated in human AF, genetically interact in mice for atrial rhythm control with unanticipated results. Surprisingly, reducing Gata4 gene dosage rescued defects caused by Tbx5 haploinsufficiency, including atrial arrhythmias, prolonged APs, abnormal ectopic cellular depolarizations, SR Ca2+ load and SERCA calcium flux. Rescue of both P-wave prolongation and ectopic cardiomyocyte depolarizations suggests that Gata4 haploinsufficiency may rescue both the vulnerable substrate and arrhythmia trigger propensity caused by Tbx5 haploinsufficiency. The identification of SERCA function as the nexus of the Tbx5/Gata4 genetic interaction suggested the more general hypothesis that calcium flux deficits caused by Tbx5 haploinsufficiency were the primary mechanism for the observed atrial rhythm disturbance. This hypothesis is supported by the ability of the reduced dose of pln, encoding a direct-binding SERCA inhibitor, to also rescue the Tbx5 heterozygote phenotype. By providing insights into the coregulation of atrial rhythm and calcium homeostasis by TBX5 and GATA4, this work illuminates complex genetic interactions that will undergird efforts toward personalized care in human rhythm control. The work also identifies SERCA calcium flux as the pathophysiologic basis of increased AF risk caused by decreased Tbx5 dose and therefore, a possible pathophysiologic mechanism germane to AF risk more generally.

We defined a calcium-handling network underlying cardiomyocyte prolonged action potentials and ectopy observed with decreased Tbx5 dose and rescued by reduced Gata4 dose. We hypothesized that normalization of SERCA expression caused normalization of AP duration and elimination of ectopic depolarization events. Consistently, reduction in Atp2a2 expression, SERCA activity, and SR Ca2+ load observed in Tbx5 haploinsufficient mice were all rescued by Gata4 haploinsufficiency (Figure 4). Recent studies investigating the cellular mechanisms of AF have suggested that abnormal atrial calcium handling contributes to AF pathogenesis, leading to trigger formation (56, 58–60). Although SR calcium content is affected by both SERCA uptake and RYR2-mediated calcium leak, our previous study suggested that Ryr2 binding was not affected in Tbx5 knockout mice, suggesting a primary role of SERCA in TBX5-dependent calcium dysfunction (61). Consistent with this model, increased SERCA activity by reduced PLN, an SERCA inhibitor, rescued the Tbx5- haploinsufficient phenotype (Figure 5). Human PLN mutations have been previously linked to AF and cardiomyopathy (62–64). Reductions in SERCA2 expression/activity or enhancement of the inhibitory effects of PLN are also hallmarks of heart failure (HF) (65, 66). Interestingly, PLN inhibition has been shown to alleviate cardiomyopathy in several animal models and improve cardiomyocyte contractility in patients with HF (67–69). Here, we show that decreased Pln dose can rescue the arrhythmogenic phenotype caused by Tbx5 haploinsufficiency, by restoring SERCA function and normalizing SR Ca2+ content (Figures 4 and 6). Zhu et al. observed rescue of heart failure and ventricular SERCA function following ablation of PLN in Tbx5vdel/+ mice (70). TBX5 therefore regulates SERCA in both the atria and ventricles for both cardiac function and rhythm. The observation that decreased Pln normalizes SR Ca2+ uptake and SERCA activity in a mouse model of AF provides a direct molecular link between calcium flux perturbations observed in HF and AF risk, which are strongly associated, regardless of genetic background. These observations suggest that PLN inhibition and more generally, the modulation of SR calcium flux, may be considered as possible therapeutic approaches for the treatment of AF in patients with HF.

Understanding of the genetic basis of cardiac rhythm control has benefited greatly from highly powered genome-wide association studies, which have identified more than 100 loci contributing to the heritability of AF. A mechanistic and actionable understanding of how the identified loci affect cardiac rhythm control requires the transition from genetic implication to functional investigation (71). The implication of the cardiogenic TFs TBX5, GATA4, and NKX2-5 by GWAS is an exciting feature, suggesting a shared transcriptional kernel in cardiac development and adult cardiac rhythm control. In the context of cardiac development, Tbx5, Gata4, and Nkx2-5 interact genetically in a synergistic fashion, their encoded TFs physically associate, and they cooperatively drive embryonic cardiac gene expression (33–44). These detailed studies provided a clear paradigm for the cooperative interaction between these TFs in adult rhythm control. Remarkably, we find that these TFs interact very differently in the adult mouse: Tbx5 and Gata4 act oppositely, and Nkx2-5 has no appreciable impact.

Understanding how TBX5 and GATA4 levels are integrated at the molecular level to afford genetic rescue of the Tbx5 mutant phenotype by reduced Gata4 activity will be essential for AF risk prediction. We identified an enhancer at Ryr2 that molecularly integrates GATA4 and TBX5 with opposite activity. Previous work has characterized multiple promoters or enhancers coregulated by TBX5 and GATA4, and in each case, positive interactions were observed, either additive or synergistic (33, 36–41). In a few cases, however, GATA4 has been reported to possess repressive molecular activity on cardiac enhancers. For example, GATA4 recruits Hdac1/2 to deacetylate specific atrioventricular canal loci, thereby repressing AV canal identity during cardiac chamber development (37). GATA4 can also cooperate with -catenin on TCFL2-enhancers in the adult heart, to maintain normal homeostasis through repression of TCFL2-driven loci (72). Therefore, GATA4 harbors repressive potential in some contexts in the heart. Here, we identified a repressive role for GATA4 at an enhancer of Ryr2, on which GATA4 opposes TBX5-dependent activation (Figure 3). We observed that TBX5 activation and GATA4 antagonism of the Ryr2 enhancer was dependent on GATA-binding sites in HL-1 cells but not HEK293T cells. One possibility for these different results is the distinct physiological conditions of these cell lines. Because HEK cells lack endogenous expression of TBX5 and GATA4, TF overexpression may overcome the effect of the binding-site mutation because TBX5 and GATA4 physically interact (33). In contrast, HL-1 cells, possessing endogenous physiologic expression of the cardiogenic kernel of TFs, including TBX5 and GATA4, may be more sensitive to the necessity of the GATA binding sites. It remains to be elucidated how GATA4 antagonizes TBX5-dependent function in the adult atrium, perhaps by recruiting repressive chromatin-remodeling enzymes or cardiac corepressors. Nonetheless, our observations provide a model for the integration of TBX5 and GATA4 dose on gene expression by the opposite modulation of single- target gene enhancers. Understanding the molecular mechanisms underlying the opposite action of TBX5 and GATA4 on cardiac gene expression will improve our understanding of the molecular basis of adult cardiac rhythm control.

This work unveiled complex genetic interactions between genes individually implicated in AF by human genetic studies. The opposite sign with which decreased Gata4 dose affected AF risk in the context of decreased Tbx5 dose indicates the importance of unveiling specific genetic interactions between genetic risk loci for understanding the combined impact of genetic variants across genomes for disease risk prediction. Human genetic studies have been underpowered for the identification of multigenic interactions to date, highlighting the importance of gene-gene interaction studies in model systems such as those included in this study. This work suggests that interaction studies between genetic risk loci will be an essential component of understanding personalized risk from genetic association studies.

Experimental design. This study was designed to investigate the oligogenic interaction of TFs that have been linked to AF by GWAS studies. We used murine models of conditional Tbx5 deletion, conditional Gata4 deletion, conditional Nkx2-5 deletion, and germline Pln heterozygosity for their similarities to human AF phenotypes. The number of mice per genotype depended on the experiments and is specified in figure legends. For animal studies, littermates were used as controls, and mice were grouped when appropriate. Endpoints for studies were selected according the phenotype of adult Tbx5-haploinsufficient mice. For single-cell electrophysiology and calcium flux measurements, 3–5 mice per genotype were analyzed, and a total of 10 cells was recorded for every mouse. All experiments, recordings, and analysis were performed in a blinded fashion. Outliers were excluded if samples/replicates were greater than 2 SDs from the population mean.

Transgenic mice.Tbx5fl/fl, Gata4fl/fl, Nkx2-5fl/fl, Pln+/–, and Rosa26CreERT2 mice have all been previously described (28, 43, 52, 73, 74). Mice were maintained on a mixed genetic background, harboring 1 copy of the Cre recombinase. All experiments involving Tbx5, Gata4, NKx2-5, and Pln heterozygotes and Gata4/Tbx5, Tbx5/Nkx2-5, Gata4/Nkx2-5, and Tbx5/Pln compound heterozygotes and Tbx5/Gata4/Nkx2-5 triple heterozygotes were performed 2 weeks after TM treatment, and age-matched littermate controls (R26CreERT2) were used for comparison. A total of 200 μl of tamoxifen (20 mg/ml in corn oil) was administered by i.p. injection for 3 consecutive days at 6 to 8 weeks of age, as previously described (17).

Telemetry ECG recordings. Ambulatory ECG studies were performed on 8- to 10-week-old mice. Mice were anesthetized using isoflurane, and telemetry transmitters (ETA-F10, Data Science International [DSI]) were implanted s.c. in the back with leads tunneled to the right upper and left lower thorax, as previously described (75). After the postimplant recovery period of 1 day, baseline recordings were collected by DSI telemetric physiological monitor system. P-wave duration, PR interval, and Poincare plots were calculated using Ponemah Physiology Platform (DSI) and custom Python script.

Intracardiac electrophysiology studies. Animals underwent catheter-based intracardiac recordings 2 weeks after receiving TM, as previously described (17). Mice were anesthetized with isoflurane, and right atrial and ventricular electrograms as well as surface electrograms were recorded using a 1.1-F octapolar catheter (EPR-800, Millar Instruments) inserted via the right jugular vein. Programmed extra-stimulation protocols and burst pacing were used to induce AT and AF in animal subjects. Programmed right atrial extra stimulation was carried out using S1 drive trains of 80 ms followed by 5 extra stimulations at 50 ms each. Burst pacing was performed by applying a series of single extra stimulus delivered at a constant pacing rate of 15–20 ppm (900–1200 bpm). Inducibility was considered positive if 2 or more series of AT and/or fibrillation was observed following burst pacing in the same animal. Fibrillation was considered noninducible if 0 or 1 cycle or irregular atrial rhythm was observed in the animal.

Echocardiography. Transthoracic echocardiography was performed 2 weeks after TM treatment under inhaled isoflurane, delivered via a nose cone, as previously described (76). Briefly, animals were imaged with a VisualSonics Vevo 770 machine (VisualSonics) using a 30-MHz high-frequency transducer. Body temperature was maintained using a heated imaging platform. Two-dimensional images were recorded in parasternal long- and short-axis projections, with guided M-mode recordings at the midventricular level in both views. M-mode echocardiographic images were obtained at the midpapillary muscle level in the parasternal short-axis view and measurement calculated at the same level from M- and/or B-mode images of long- or short-axis view. Left ventricular dimensions in both diastole and systole were measured from at least 3 different beats from each projection and averaged to calculate the left ventricular ejection fraction.

Calcium flux measurement. Single-cell atrial cardiomyocytes were isolated by Langendorff perfusion with 2 mg/ml of Collagenase Type 2 at 5 ml/min. Atrial myocytes were plated on laminin-coated glass-bottom dishes and incubated at room temperature for 30 minutes prior to incubation with Fluo-4 AM (Molecular Probes/Invitrogen). Cells were then incubated with 10-μM Fluo-4 AM for 1 hour in normal Tyrode’s solution containing (in mM): 140 NaCl, 4 KCl, 10 glucose, 10 HEPES, and 1 MgCl2, 1 CaCl2, with pH 7.4 using NaOH, followed by a 10-minute perfusion wash with pre-warmed Tyrode’s solution. SERCA and NCX measurements were performed using an Olympus microscope with a x20 objective lens, a LAMBDA DG-4 power source with 488-nm excitation and 515-nm emission filters and a PMT (Microphotometer) to record whole-cell signal, with electrical field stimulation (Grass stimulator; Astro-Med) at 1 Hz. SERCA and NCX activities were measured as follows: 10-mM caffeine- containing solution was applied in the absence of extracellular sodium and the cells returned to normal Tyrode’s solution at the end of the recording. In the presence of sodium-free caffeine Tyrode’s solution, the intracellular calcium remains elevated, and the peak value is a measure of SR calcium load that was released into the cytosol.

Diastolic calcium measurements. Atrial myocytes were isolated by Langendorff perfusion, plated on laminin-coated glass-bottom dishes, and incubated at room temperature for 30 minutes before staining with Fura-2 AM. Cells were then incubated with 1-μM Fura-2 AM for 10 minutes in normal Tyrode’s solution. Diastolic calcium measurements were performed at a fluorescence emission of 510 nm and recorded using an Olympus IX81 Inverted Widefield Microscope. [Ca]i was calculated by the ratio of emissions following excitation at 340 nm and 380 nm.

Relative luciferase assays. HEK293T and HL-1 cells were cotransfected as described previously (17). The Atp2a2 and Ryr2 cis-regulatory elements were amplified from C57/B6 mouse genomic DNA. The sequence was confirmed by sequencing and then cloned into the pGL4.23 enhancer luciferase response vector with a minimal promoter. Mutagenesis of the GATA4-binding motifs was performed by PCR cloning, confirmed by sequencing and then cloned into the pGL4.23 vector. Cells were co-transfected with the luciferase response vector and pRL control using lipofectamine 3000, then lysed and assayed following 48 hours using the Dual-Luciferase Reporter Assay system (Promega).

Quantitative real-time PCR. Total RNA was extracted from the left atrial wall of 8- to 10-week-old adult mice (2 weeks after receiving TM) using Trizol (Invitrogen) combined with RNeasy mini-kit (Qiagen), according to the manufacturer’s instructions. First-strand cDNA was synthesized using the qScript cDNA synthesis kit (Quanta) according to the manufacturer’s protocol. Gene expression was assayed using the Power SYBR Green PCR Master Mix (Applied Biosystems) and run on an Applied Biosystems AB7500 machine in 96-well plates. Relative fold changes were calculated using the comparative threshold cycle method (2–ΔCt) using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as an internal control. Primer sequences used in this study are listed in Supplemental Table 3.

Statistics. All data are represented as means ± SEM. For comparison of conscious ECG parameters, action potential duration (APD50 and APD90), SR load, and SERCA activity, a 1-way ANOVA followed by post-hoc Tukey analysis was used to test significance. For gene-expression studies and in vitro luciferase assays, a 1-way ANOVA followed by internal Student’s t test was used to test significance. AF inducibility and triggered activity (from AP measurements) counts were analyzed with a Fisher’s exact test (2-tailed).

Study approval. All animal experiments were performed in accordance with national and institutional guidelines and were approved by the University of Chicago Institutional Animal Care and Use Committee.

BL and WD were involved in experimental design, execution and analysis, wrote the manuscript, and performed statistical analysis. BL performed and analyzed ambulatory ECG, whole-animal electrophysiology studies, and gene expression. WD performed and analyzed calcium flux measurements. LT performed and analyzed single-cell electrophysiology experiments. SL, KMS, and MG performed and analyzed luciferase assays. MTB performed and analyzed intracardiac electrophysiology studies. CRW performed and analyzed single-cell electrophysiology experiments and wrote the manuscript. IPM was involved in the design, execution, and analysis of experiments and wrote the manuscript.

This work was supported by the NIH (R01 HL148719 and R01 HL147571 to IPM; K08HL129073 to MTB), the American Heart Association (7CSA33610126 to IPM) and the Leducq Foundation (to IPM). We thank Evangelia Kranias for the Phospholamban mice (NIH R01 HL26057). This research was supported in part by the NIH through resources provided by the Computation Institute and the Biological Sciences Division of the University of Chicago and Argonne National Laboratory, under grant 1S10OD018495-01.